Cholesterol and Caveolin-1 Modulate Lateral Difussion, Expression and Function of the Novel Slow Channel Congenital Myasthenia Syndrome α418w Nachr Mutation At Lipid Rafts
Oyola Cintrón, Jessica
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The nicotinic acetylcholine receptor (nAChR) is part of the Cys-loop family of ligand gated ion channels. In recent years there has been a substantial increase in the number of studies that focus their attention on the lipid-protein interface of the nAChR. However, important concepts regarding this complex network of lipids and proteins remains to be completely understood. Thus, there is a need to identify and gain insight into the mechanism through which lipid-protein interactions regulate nAChR function and dynamics. This dissertation aims to define the molecular basis for the regulation of the functional pool of nAChRs via membrane cholesterol microdomains. Its objective is to gain insight into the mechanism by which lateral trafficking and segregation into specialized raft membrane microdomains regulates the activable pool of nAChRs. Specifically, we aim to determine if an inhibition of macroscopic peak currents in wholecell recordings upon a physiologically relevant cholesterol enrichment correlates with a reduction in the mobile fraction of nAChRs by means of Fluorescent Recovery After Photobleaching (FRAP) experiments of GFP-encoding Mus musculus nAChRs in HEK 293 cells. This approach reveals an interplay between cholesterol and CAV-1 that provides the molecular basis for modulating the function and dynamics of the cholesterolsensitive αC418W nAChR, the first lipid-exposed mutation identified in a patient that causes Slow Channel Congenital Myasthenia Syndrome (SCCMS). The contributions of this dissertation to the field are significant as it provides novel information about the regulation of nAChR function and dynamics by cholesterol and CAV-1; thus having an impact on potential therapies for diseases involving disruption of the function and dynamics of membrane proteins. In addition, the measurement of lateral diffusion of the αC418W mutant nAChR in cholesterol-enriched and cholesterol-depleted conditions represents the first attempt to visualize αC418W nAChR trafficking between raft membrane microdomains in vivo. The principles that govern the regulation of AChR function, dynamics and trafficking across the membrane are expected to be relevant to other important receptor systems, ion channels and membrane proteins. Therefore, the findings of this dissertation will have an impact on potential therapies for diseases involving disruption of the function and dynamics of membrane proteins. Our findings provide novel information about the interactions between the nAChR and the components of the lipid-protein interface, a complex network that will continue to be the focus of novel therapeutics for diseases involving membrane proteins.